I. Gene Therapy
Development of gene therapy techniques is approaching clinical realization for the treatment of neoplastic and metabolic diseases. There remains substantial need for improvement both in the vector delivery systems for delivery of the transgene to target tissues, and the identification of genes most effective for anti-tumor therapy.
Vectors for carrying genes may be viral or non-viral. For example, replication-deficient retroviral vectors can efficiently transfect dividing cells. Local intratumoral injection of retroviruses that contain a thymidine kinase transgene has been used successfully to affect regression of gliomas (Culver et al, Science, 2:1550-1552 (1992)). Unlike retroviral vectors, adenoviral vectors can also transfect non-dividing cells, and their ability to cause insertional mutagenesis is greatly reduced. However, they can have the undesirable potential to activate the immune system in humans (Crystal, Science, 270:404-410, (1995). Attempts are underway to minimize the immunogenicity of the adenoviral vectors.
Non-viral vectors of DNA include primarily liposomes, peptides, proteins and polymers (Ledley, Current Opinion in Biotechnology, 5:626-636 (1994)). Of these, liposomes are currently the most common non-viral vectors of DNA. The major advantage of liposomes over retroviruses is that DNA is not incorporated into the genome, and unlike adenoviral vectors, they are not immunogenic. However, the major limitation of liposomes is that they are not as efficient as viral vectors in transfecting many cell types. Until recently, their medical utility was limited by their rapid uptake by phagocytic cells. Interest in liposomes as a vector has been increased by two technological advances. First, stearically stabilized (Stealth) liposomes have been developed which are more non-reactive and are not readily taken up by the reticuloendothelial system (RES). Stealth liposomes are composed of lipids rich in oxygen in their head group (ethylene glycol or glycolipids) which provide a stearic barrier outside of the membrane. As a result, Stealth liposomes remain in the blood for up to 100 times longer than conventional liposomes, and can thus increase pharmacological efficacy (Papahajopoulos, In: Stealth Liposomes, Ed., Lasic et al, CRC Press (1995); and Lasic et al, Science, 267:1275-76 (1995)). However, stealth liposomes are still not particularly efficient in transfection of cells or as vectors for DNA.
The second significant advance in liposome technology has been the use of cationic liposomes complexed to negatively-charged DNA. Cationic liposomes can condense DNA, and increase transfection yields several orders of magnitude. In the cationic liposome:DNA complex, the nucleic acids or oligonucleotides are not encapsulated, but are simply complexed with small unilamellar vesicles by electrostatic interactions. The exact nature of the cationic liposome:DNA complex is not fully known, but intricate topological rearrangements of the cationic liposome:DNA complex may occur, including DNA condensation, liposome aggregation, and fusion. This supramolecular complex can be added to cells in vitro, injected parenterally, or aerosolized for pulmonary applications (Lasic et al, Science, 267:1275-1276 (1995)). Further, the intravenous injection into mice of high concentrations of the CAT gene (100 .mu.g or greater) complexed with cationic liposomes has been found to result in 40% transfection efficiency of well vascularized tissues, such as the spleen (Zhu et al, Science, 261:209-211 (1993)). Notwithstanding these advances, a major challenge of gene therapy remains the systemic delivery of transgenes to the tumor or peritumoral area that will effectively decrease the size of primary tumors and their metastases. Unlike the spleen and bone marrow, which are highly vascular and have a high capacity to filter macromolecules from the blood stream, most organs and tumors do not have this capacity, and the transfection efficiency of these tissues with liposomes is low (Marshall, Science, 269:1051-1055 (1995)). In addition, another limitation of cationic liposome:DNA complexes is that their 1/2 life in the blood stream is normally less than one hour (Allen et al, In: Liposome Technology-Vol. III, Ed., Gregoriadis G et al, CRC Press (1993); Li and Huang, J. of Liposome Research, 6:589 (1996). Sufficient transfection of the target cell by vectors carrying therapeutic genes has thus far been the rate-limiting step in gene therapy.
II. Tumor Suppressor Genes
Tumor suppressor genes are well-known in the art, and include the p53 gene (Baker et al, Science, 249:912-915 (1990)), the p21 gene (El-Deiry et al, Cell, 75:817-825 (1993); and Harper et al, Cell, 75:805-816 (1993)), and the rb gene (Bookstein et al, Science, 247:712-715 (1990)).
Mutations in the tumor suppressor gene p53 are known to occur in over 50% of human tumors, including metastatic breast cancer. Various groups have found that reintroduction of the wild-type P53 by mediated transfer of a single copy of the p53 transgene into a variety of tumor cells, including breast cancer cells, results in a decrease in growth rate and/or attenuated tumor development once those transfected cells were implanted into nude mice (Wang et al, Oncogene, 8:279-288 (1993); Baker et al, Science, 249:912-915 (1990)); Bookstein et al, Science, 247:712-715 (1990); Cheng et al, Cancer Res., 52:222-226 (1992); Isaacs et al, Cancer Res., 51:4716-4720 (1991); Diller et al, Mol. Cell. Biol., 10:5772-5781 (1990); Chen et al, Oncogene, 6:1799-1805 (1991); and Zou et al, Science, 263:526-529 (1994)). In addition, intratracheal injection of a retrovirus containing the p53 transgene has been shown to significantly inhibit the growth of lung tumors (Fujiwara et al, J. Natl. Cancer. Inst., 86:1458-1462 (1994)).
Systemic intravenous administration of a .beta.-actin promoter-containing vector containing the p53 coding sequence complexed to cationic liposomes has been found to affect the tumor growth of a malignant line of breast cancer cells injected into nude mice (Lesoon-Wood et al, Proc. Am. Ass. Cancer Res., 36:421 (1995); and Lesoon-Wood et al, Human Gene Ther., 6:39-406 (1995)). Of the 15 tumors treated in this study, four of these tumors did not respond to treatment. Because of the unresponsiveness of these tumors, new therapies were sought in the present invention to more effectively decrease the size of these tumors.
p53 coordinates multiple responses to DNA damage. DNA damage results in an increase in the level of the p53 protein. Following DNA damage, an important function of wild-type p53 function is to control the progression of cells from G1 to S phase. Recently, several groups have found that p53 transcriptionally activates a p21 kd protein (also known as WAF1 or CIP1), an inhibitor of cyclin-dependent kinases (CDKs) (El-Deiry et al, supra; and Harper et al, supra). Inhibition of CDK activity is thought to block the release of the transcription factor E2F, and related transcription factors from the retinoblastoma protein RB, with consequent failure to activate transcription of genes required for S phase entry (Harper et al, supra; and Xiong et al, Nature, 366:701-704 (1993)). Evidence consistent with the model that pRb is a downstream effector of p53-induced GI arrest has recently been reported (Dulic et al, Cell, 76:1013-1023 (1994)). Thus, p53 regulates cell cycle through two proteins: p21 and rb.
III. Anti-Angiogenic Proteins
Proteins with anti-angiogenic activities are well-known and include: thrombospondin I (Kosfeld et al, J. Biol. Chem., 267:16230-16236 (1993); Tolsma et al, J. Cell Biol., 122:497-511 (1993); and Dameron et al, Science, 265:1582-1584 (1995)), IL-12 (Voest et al, J. Natl. Cancer Inst.,87:581-586 (1995)), protamine (Ingber et al, Nature, 348:555-557 (1990)), angiostatin (O'Reilly et al, Cell, 79:315-328 (1994)), laminin (Sakamoto et al, Cancer Res., 5:903-906 (1991)), endostatin (O'Reilly et al., Cell, 88:277-285 (1997)), and a prolactin fragment (Clapp et al, Endocrinol., 133:1292-1299 (1993)). In addition, several anti-angiogenic peptides have been isolated from these proteins (Maione et al, Science, 247:77-79 (1990); Woltering et al, J. Surg. Res., 50:245-251 (1991); and Eijan et al, Mol. Biother., 3:38-40 (1991)).
Thrombospondin I (hereinafter "TSPI") is a large trimeric glycoprotein composed of three identical 180 kd subunits (Lahav et al, Semin. Thromb. Hemostasis, 13:352-360 (1987)) linked by disulfide bonds (Lawer et al, J. Cell Biol., 103:1635-1648 (1986); and Lahav et al, Eur. J. Biochem., 145:151-156 (1984)). The majority of anti-angiogenic activity is found in the central stalk region of this protein (Tolsma et al, supra). There are at least two different structural domains within this central stalk region that inhibit neovascularization (Tolsma et al, supra).
Besides TSPI, there are six other proteins (fibronectin, laminin, platelet factor-4, angiostatin, endostatin and prolactin fragment) in which peptides have been isolated that inhibit angiogenesis. In addition, the dominant negative fragment of FlK1 and analogues of the peptide somatostatin are known to inhibit angiogenesis.
Fibronectin (FN) is a major surface component of many normal cells, as well as a potent cell spreading factor. During transformation, the loss of cellular FN has been observed. Furthermore, the addition of fibronectin to transformed cells restores the normal phenotype. It has been found that either heparin-binding or cell-adhesion fragments from FN can inhibit experimental metastasis, suggesting that cell surface proteolyglycans are important in mediating the adhesion of metastatic tumor cells (McCarthy et al, J. Natl. Cancer Inst., 80:108-116 (1988)). It has also been found that FN and one of its peptides inhibits in vivo angiogenesis (Eijan et al, Mol. Biother., 3:38-40 (1991)).
Laminin is a major component of the basement membrane, and is known to have several biologically active sites that bind to endothelial and tumor cells. Laminin is a cruciform molecule that is composed of three chains, an A Chain and two B chains. Several sites in laminin have been identified as cell binding domains. These sites promote cellular activities in vitro, such as cell spreading, migration, and cell differentiation. Two peptides from two sites of the laminin B1 chain are known to inhibit angiogenesis (Grant et al, Path. Res. Pract., 190:854-863 (1994)).
Platelet factor-4 (PF4) is a platelet .alpha.-granule protein originally characterized by its high affinity for heparin. The protein is released from platelets during aggregation as a high molecular weight complex of a tetramer of the PF4 polypeptide and chondroitin sulfate, which dissociates at high ionic strength. PF4 has several biological properties including immunosuppression, chemotactic activity for neutrophils and monocytes as well as for fibroblasts, inhibition of bone resorption, and inhibition of angiogenesis. The angiostatic properties of human PF4 are associated with the carboxyl-terminal, heparin binding region of the molecule. A 12 amino acid synthetic peptide derived therefrom has been discovered to have marked angiostatic affects (Maione et al, Science, 247:77-79 (1990)).
Endostatin is a 20 kDa protein fragment of collagen XVIII. It has recently been found to be a potent inhibitor of tumor angiogenesis and tumor growth (O'Reilly et al., Cell, 88, 277-285, 1997).
Although somatostatin is not a protein, it is a naturally-occurring cyclic 14 amino acid peptide whose most-recognized function is the inhibition of growth hormone (GH) secretion. Somatostatin is widely distributed in the brain, in which it fulfills a neuromodulatory role, and in several organs of the gastrointestinal tract, where it can act as a paracrine factor or as a true circulating factor. The role played by the neuropeptide somatostatin, also known as somatotropin release inhibitory factor (SRIF), in human cancer is not well understood. Recent investigations involving somatostatin receptors in normal and neoplastic human tissues suggest that the action is complex, and involves both direct and indirect mechanisms. One of the anti-tumor mechanisms of these synthetic somatostatin analogues may be an anti-angiogenic effect (Woltering et al, J. Surg. Res., 50:245-50 (1990)). In a recent study, the ability of native somatostatin and nine somatostatin analogues to inhibit angiogenesis were evaluated. The most potent somatostatin analogue was found to be approximately twice as potent as the naturally-occurring somatostatin (Barrie et al, J. Surg. Res., 55:446-50 (1993)).
Angiostatin is a 38 kDa polypeptide fragment of plasminogen. Whereas plasminogen has no fibrinogenic activity, angiostatin has marked angiogenic activity (O'Rielly MS, et al Cell, 79:315-28 (1994)). Angiostatin was isolated when it was observed that the primary tumor suppressed metastases. That is, when the primary tumor was removed, the metastases grew. Administration of angiostatin blocks neo-vascularization and growth of metastases.
The Flk1 receptor is a receptor for vascular endothelial growth factor (VEGF). FlK-1 is exclusively expressed on the surface of the endothelial cells. Once VEGF binds to the receptor, the Flk-1 receptor then homodimerizes to stimulate the endothelial cell to divide. If a mutant receptor of Flk-1 is transfected into the endothelial cells, the mutant receptor dimerizes with the wild-type Flk-1 receptor. In this endothelial transfected with the mutant Flk-1 receptor, VEGF is unable to stimulate the endothelial cells to divide. Co-administration of a retrovirus carrying the Flk-1 cDNA (Millauer B. et a., Nature, 367, 1994) inhibits tumor growth. This emphasizes that the receptor plays a critical role in the angiogenesis of solid tumors.
Finally, a 16 kd fragment of prolactin has been found to be antiangiogenic. Similar to plasminogen, prolactin is not anti-angiogenic but the prolactin fragment is a potent in vivo and in vitro inhibitor of angiogenesis (Clapp C. et al. Endocrinology. 133:1292-1299 (1993).
Despite the evidence that anti-angiogenic peptides can be useful anti-tumor agents, and interest in targeting genes toward the vasculature, there have been no published reports on effective in vivo gene therapy regimens utilizing anti-angiogenic DNA sequences.
The only transfected antiangiogenic gene that has been shown to inhibit tumor growth is full length thrombospondin I. In that study (Weinstat-Saslow et al, Cancer Research 54, 6504-6511, (1994)) tumor cells that expressed 15-fold higher levels of the thrombospondin I in vitro than baseline cells were implanted into mice. This transfected full length thrombospondin I was secreted from the tumor cells, and effectively reduced the tumor by 60%. Thus, this study determined that transfection of 100% of the tumor cells with a highly expressed and secreted antiangiogenic gene was able to reduce tumor size.